U.S. patent application number 13/878358 was filed with the patent office on 2014-01-02 for reversibly water-soluble nanocrystals.
This patent application is currently assigned to EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY. The applicant listed for this patent is Lu Liu, Xinhua Zhong. Invention is credited to Lu Liu, Xinhua Zhong.
Application Number | 20140004562 13/878358 |
Document ID | / |
Family ID | 48798526 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140004562 |
Kind Code |
A1 |
Zhong; Xinhua ; et
al. |
January 2, 2014 |
REVERSIBLY WATER-SOLUBLE NANOCRYSTALS
Abstract
A general, facile, and reversible nanocrystal (NCs) phase
transfer protocol via ligand exchange using nucleotides and/or
nucleosides is provided to generate reversibly water-soluble
nanocrystals. This phase transfer strategy can be employed on a
wide variety of chemically synthesized nanostructured materials
including semiconductors, metal oxides and noble metals with
different sizes and shapes. The nucleotide/nucleoside-capped
nanocrystals can disperse homogeneously in aqueous or alcohol media
retaining, for example, high photoluminescence quantum yields. The
disclosed water-soluble nanocrystals have excellent colloidal and
photoluminescent stability independent on the pH and ionic
strength, minimal hydrodynamic size, and are stable in cells and
suitable for in vitro cell labeling, cell tracking, and other
bioimaging applications.
Inventors: |
Zhong; Xinhua; (Shanghai,
CN) ; Liu; Lu; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Zhong; Xinhua
Liu; Lu |
Shanghai
Shanghai |
|
CN
CN |
|
|
Assignee: |
EAST CHINA UNIVERSITY OF SCIENCE
AND TECHNOLOGY
Shanghai
CN
|
Family ID: |
48798526 |
Appl. No.: |
13/878358 |
Filed: |
January 20, 2012 |
PCT Filed: |
January 20, 2012 |
PCT NO: |
PCT/CN2012/070659 |
371 Date: |
April 8, 2013 |
Current U.S.
Class: |
435/40.5 ;
435/366; 435/370 |
Current CPC
Class: |
C07H 19/20 20130101;
B82Y 30/00 20130101; C07H 19/10 20130101; G01N 33/5005
20130101 |
Class at
Publication: |
435/40.5 ;
435/366; 435/370 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A reversibly water-soluble nanocrystal comprising a core
nanocrystal capped by a plurality of nucleotides, a plurality of
nucleosides or a combination thereof.
2. The reversibly water-soluble nanocrystal of claim 1, wherein the
core nanocrystal is a quantum dot, a nanorod, a metal oxide
nanocube, or a noble metal nanodot.
3. The reversibly water-soluble nanocrystal of claim 1, wherein the
core nanocrystal comprises a quantum dot having a core/shell/shell
structure.
4.-6. (canceled)
7. The reversibly water-soluble nanocrystal of claim 1, wherein the
plurality of nucleotides comprises adenosine 5'-monophosphate,
guanosine 5'-monophosphate, cytidine 5'-monophosphate, uridine
5'-monophosphate, thymidine 5'-monophosphate, or combinations
thereof, and wherein the plurality of nucleosides comprises
adenosine, guanosine, cytidine, uridine, thymidine, inosine, or
combinations thereof.
8. The reversibly water-soluble nanocrystal of claim 1, wherein the
plurality of nucleotides comprises adenosine 5'-diphosphate,
guanosine 5'-diphosphate, cytidine 5'-diphosphate, uridine 5'
-diphosphate, thymidine 5' -diphosphate, adenosine 5'
-triphosphate, guanosine 5'-triphosphate, cytidine 5'-triphosphate,
uridine 5'-triphosphate, thymidine 5'-triphosphate, or combinations
thereof.
9. The reversibly water-soluble nanocrystal of claim 1, wherein the
plurality of nucleotides comprises deoxynucelotides and wherein the
plurality of nucleosides comprises deoxynucleosides.
10. The reversibly water-soluble nanocrystal of claim 1, further
characterized by being soluble and stable in water for at least
about sixty days without substantial aggregation or
precipitation.
11. The reversibly water-soluble nanocrystal of claim 1, further
characterized by retaining substantially the same size, morphology,
and size distribution when dispersed in water as compared to the
core nanocrystal when capped by hydrophobic ligands and dispersed
in hydrophobic media.
12. The reversibly water-soluble nanocrystal of claim 1, further
characterized by retaining substantially the same luminescence
brightness and colloidal stability when dispersed in water as when
dispersed in a hydrophobic media.
13. The reversibly water-soluble nanocrystal of claim 1, wherein
the plurality of nucleotides, the plurality of nucleosides or the
combination thereof that have capped the core nanocrystal form a
layer that is equal to or less than about five nanometers
thick.
14.-16. (canceled)
17. The reversibly water-soluble nanocrystal of claim 1, further
characterized in that the water-solubility may be reversed by
displacing the plurality of nucleotides and/or nucleotides that
have capped the core nanocrystal with a plurality of hydrophobic
capping ligands.
18. (canceled)
19. The reversibly water-soluble nanocrystal of claim, further
characterized in that the conversion between water-solubility and
water-insolubility may be carried out for at least ten cycles
without significantly altering the performance characteristics of
the nanocrystal.
20.-25. (canceled)
26. The reversibly water-soluble nanocrystal of claim 1, further
characterized by retaining substantially the same luminescence
brightness and colloidal stability within a biological cell when
compared to outside a cell in aqueous media.
27.-33. (canceled)
34. A method for producing reversibly water-soluble nanocrystals,
the method comprising the steps of: (i) providing water insoluble
nanocrystals comprising core nanocrystals capped by a plurality of
initial hydrophobic ligands; and (ii) contacting the water
insoluble nanocrystals of step (i) with a plurality of nucleotides,
a plurality of nucleosides, or a combination thereof, thereby
replacing the initial hydrophobic ligands that cap the core
nanocrystal with a cap of nucleotides, nucleosides, or a
combination thereof, thus rendering the core nanocrystal reversibly
water-soluble.
35. The method of claim 34, wherein the core nanocrystal is
selected from the group consisting of a quantum dot, a nanorod, a
metal oxide nanocube, and a noble metal nanodot.
36. The method of claim 34, wherein the plurality of nucleotides
comprises adenosine 5' -monophosphate, guano sine 5'
-monophosphate, cytidine 5' -monophosphate, uridine
5'-monophosphate, thymidine 5'-monophosphate, or combinations
thereof, and wherein the plurality of nucleosides comprises
adenosine, guanosine, cytidine, uridine, thymidine, inosine, or
combinations thereof.
37. The method of claim 34, wherein the plurality of nucleotides
comprises adenosine 5'-diphosphate, guanosine 5'-diphosphate,
cytidine 5'-diphosphate, uridine 5'-diphosphate, thymidine
5'-diphosphate, adenosine 5'-triphosphate, guanosine
5'-triphosphate, cytidine 5'-triphosphate, uridine 5'-triphosphate,
thymidine 5'-triphosphate, or combinations thereof.
38. A method for reversibly transferring nanocrystals between a
hydrophobic solution and a hydrophilic solution, the method
comprising: (i) providing a hydrophobic solution comprising core
nanocrystals capped by a plurality of hydrophobic ligands; (ii)
mixing the hydrophobic solution with a solution comprising a
plurality of nucleotides and/or nucleosides in order to replace the
hydrophobic ligands capping the core nanocrystal with the
nucleotides and/or nucleosides, thereby producing a
ligand-exchanged solution comprising nucleotide- and/or
nucleoside-capped nanocrystals; and (iii) mixing the
ligand-exchanged solution with a hydrophilic solution, whereby the
nucleotide- and/or nucleoside-capped nanocrystals transfer from the
hydrophobic, ligand-exchanged solution to the hydrophilic
solution.
39.-40. (canceled)
41. The method of claim 38, wherein the core nanocrystal comprises
a quantum dot, a nanorod, a metal oxide nanocube, a noble metal
nanodot, or combinations thereof.
42. The method of claim 38, wherein the plurality of nucleotides
comprises adenosine 5' -monophosphate, guano sine 5'
-monophosphate, cytidine 5' -monophosphate, uridine
5'-monophosphate, thymidine 5'-monophosphate, or combinations
thereof, and wherein the plurality of nucleosides comprises
adenosine, guanosine, cytidine, uridine, thymidine, inosine, or
combinations thereof.
43. The method of claim 38, wherein the plurality of nucleotides
comprises adenosine 5'-diphosphate, guanosine 5'-diphosphate,
cytidine 5'-diphosphate, uridine 5' -diphosphate, thymidine
5'-diphosphate, adenosine 5' -triphosphate, guanosine
5'-triphosphate, cytidine 5'-triphosphate, uridine 5'-triphosphate,
thymidine 5'-triphosphate, or combinations thereof.
44. The method of claim 38, further comprising: (iv) mixing the
hydrophilic solution with (a) a solution comprising a plurality of
hydrophobic ligands and (b) a secondary hydrophobic solution in
order to replace the nucleotides and/or nucleosides capping the
core nanocrystal with the hydrophobic ligands, thereby producing a
secondary ligand-exchanged solution comprising hydrophobic
ligand-capped nanocrystals, whereby the hydrophobic ligand-capped
nanocrystals transfer from the hydrophilic solution to the
secondary hydrophobic solution.
45. The method of claim 44, wherein the hydrophobic ligands
comprise organic amine ligands.
46.-47. (canceled)
48. A composition comprising a biological reagent coupled to a
reversibly water-soluble nanocrystal comprising a core nanocrystal
capped by a plurality of nucleotides, a plurality of nucleosides,
or a combination thereof.
49. The composition of claim 48, wherein the biological reagent
comprises an antibody, a nucleic acid probe, an enzyme substrate, a
binding protein, or combinations thereof.
50. (canceled)
51. A method for labeling a biological cell or molecular component
of such cell, the method comprising the step of: contacting the
cell or molecular component with a composition comprising a
biological reagent coupled to a reversibly water-soluble
nanocrystal comprising a core nanocrystal capped by a plurality of
nucleotides, a plurality of nucleosides, or a combination
thereof.
52.-54. (canceled)
55. A biological cell or molecular component of such cell labeled
with a composition comprising a biological reagent coupled to a
reversibly water-soluble nanocrystal comprising a core nanocrystal
capped by a plurality of nucleotides, a plurality of nucleosides,
or a combination thereof.
56. The biological cell or molecular component of such cell of
claim 55, wherein the biological cell is a cancer cell.
57. The biological cell or molecular component of such cell of
claim 55, wherein the molecular component of such cell is a nuclear
membrane.
Description
BACKGROUND
[0001] During the past decade, colloidal nanocrystals (NCs) have
found numerous applications in optoelectronics, photovoltaic cells,
catalysis, and biotechnology, due to their distinguished size- and
shape-dependent properties. The innately high surface
area-to-volume ratio of NCs results in the surface containing a
large fraction of unsaturated atoms. For stabilization and
functionalization purposes, organic surfactants are typically
adsorbed onto the surface of NCs as a "cap" to passivate the
dangling bonds. The capping organic ligands/surfactants stabilize
the NCs dispersion and determine the physicochemical properties of
the nanoparticles, such as the hydrodynamic size, toxicity, charge
and intermolecular/interparticle interactions. The NC-organic
surfactant interface plays an important role in NC structure and
optoelectronics, therefore the ability to engineer surface
properties of NCs is important for various applications.
[0002] Most chemically synthetic routes to high-quality NCs
predominantly employ carboxylic acids, amines, or phosphine oxides
with long hydrocarbon chains as capping ligands, which sterically
stabilize NCs in hydrophobic solvents. However, the presence of
such bulky capping molecules creates an insulating bather around
each NC and blocks the access of molecular species to the NC
surface, which is detrimental for electronic and catalytic
applications. In addition, biological applications, such as cell-
or organelle-staining, generally require NCs to be water-soluble
and biocompatible, for which traditional hydrophobic, ligand-capped
NCs are not suitable.
[0003] To address such specific applications, transfer of NCs from
a hydrophobic environment to a hydrophilic one, or vice versa, is
required in order to maximize the respective advantages of these
environments. This makes phase transfer an important aspect in the
functionalization and application of nanostructured materials.
Typical phase transfer of NCs is achieved by replacing the original
ligands with specifically designed molecules through a
ligand-exchange process, or by cross-linking of NCs with a shell of
silanols, or amphiphilic copolymers. Although surface modification
based on ligand-exchange reactions has been actively explored in
various NC systems, a generalized and efficient strategy is far
from developed. Thiol-functionalized carboxylic acid ligands such
as mercaptopropionic acid (MPA), dihydrolipoic acid (DHLA) are most
often been employed for ligand exchange reaction. The
thiol-functionalized ligands capped NCs are not stable enough due
to the facile oxidization feature of the thiol group. In addition,
due to the strong binding ability of the thiol group, this
ligand-exchange process is typically irreversible, making it
difficult to further functionalize NCs. To date, all reported
reversible phase transfer approaches suffer from one or more of the
following problems: i) the phase transfer process will deteriorate
physicochemical properties of NCs, such as colloidal stability and
optical properties; ii) the process only works for certain NC
systems; iii) NCs may be dispersed in either polar or nonpolar
solvent, but not truly and repeatedly reversible between aqueous
and organic media; iv) the phase transfer reagents require tedious
synthesis and are expensive.
[0004] Accordingly, there remains a need to develop a generalized,
facilely reversible phase transfer methodology and materials for
functionalized NCs between aqueous and organic phases.
SUMMARY
[0005] A general ligand-exchange methodology is provided using
nucleotides and/or nucleosides to replace the initial hydrophobic
ligands (with long hydrocarbon tails) on the surface of NCs, thus
rendering the initial oil-soluble NCs reversibly water-soluble. A
reversibly water-soluble nanocrystal comprising a core nanocrystal
capped by a plurality of nucleotides, a plurality of nucleosides,
or a combination thereof is provided. The disclosed methodology is
suitable for a variety of NC systems such as semiconductor, metal
oxide, and noble metal of different sizes and shapes. The method
may, for example, be employed with luminescent quantum dots (QDs),
since rendering the as-synthesized QDs water-soluble and
biocompatible, while preserving the high luminescent brightness and
maintaining colloidal and chemical stability, is of great
importance for biological applications. The disclosed nucleotides-
and/or nucleosides-capped QDs, after phase transfer, endow QDs with
excellent fluorescent and colloidal stability independent on the pH
and ionic strength, minimal hydrodynamic size, and are thus
suitable potential fluorophores in biomedical imaging applications.
The disclosed reversibly water-soluble NCs can be readily further
functionalized by hydrophobic ligands via a secondary
ligand-exchange reaction, allowing fully reversible phase transfer
and surface functionalization of NCs. Also provided are methods of
using the disclosed reversibly water-soluble nanocrystals to label
a biological cell or molecular component of such cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIGS. 1A-D--are transmission electron microscopy (TEM)
images of initial oil-soluble NCs before phase transfer: (A)
CdSe/CdS/ZnS QDs, (B) Bi.sub.2S.sub.3 nanorods, (C) MnO nano-cubes,
and (D) Au nanadots.
[0007] FIGS. 2A-B--are luminescence images of initial oil-soluble
QDs in hexane, and alcohol-soluble QDs dispersed in methanol and
ethylene glycol (EG) after phase transfer with use of AMP
lamination by room light (A) and UV light (B).
[0008] FIGS. 3A-D--are TEM images of AMP-modified NCs dispersed in
water derived from the initial oil-soluble ones via phase transfer:
(A) CdSe/CdS/ZnS QDs (6.0 nm), (B) Bi2S3 nanorods (8 nm.times.46
nm), (C) MnO nano-cubes (23 nm), and (D) Au nanodots (7.5 nm). The
insets show photographs of the initial OAm-capped NCs dispersed in
hexane (left) and the resulting AMP-capped NCs dispersed in water
(right) after phase transfer.
[0009] FIGS. 4A-D--are luminescence images of initial oil-soluble
QDs solutions in hexane, and water-soluble QDs in water under UV
light irradiation after phase transfer with use of AMP (a) and
adenosine (b) as phase transfer reagents, respectively. UV-vis (c)
and PL (d, .lamda..sub.ex=360 nm) spectra of initial hydrophobic
CdSe/CdS/ZnS QDs in hexane solutions (solid curves), and the
corresponding AMP-capped QDs in aqueous media (dashed curves).
Note: the PL intensities of all oil-soluble QDs were normalized to
100, and the PL spectra of the corresponding water-soluble QDs
after phase transfer were recorded under the same condition as for
oil-soluble QDs.
[0010] FIGS. 5A-B--are TEM images of initial oil-soluble QDs (A)
and corresponding water-soluble ones after phase transfer with use
of AMP (B). Inset: Dynamic light-scattering histograms of
corresponding water-soluble QDs with average hydrodynamic diameter
of 7.1 nm.
[0011] FIGS. 6A-C--are Fourier-transform infrared (FTIR) spectra of
initial oil-soluble OAm-QDs (A), AMP-QDs after phase transfer (B),
and free sodium Na.sub.2-AMP (C).
[0012] FIG. 7--is an FTIR spectrum of the OAm-capped QDs recovered
from the water-soluble AMP-QDs by phase-transfer back to the
hydrophobic phase.
[0013] FIGS. 8A-C--are (A) a schematic illustration of the
reversible phase transfer process of QDs sample dispersed in hexane
and water. (B) Relative PL intensity variation of oil- and
water-soluble QDs in each cycle of phase transfer process. (C)
Representative PL emission spectra of the initial oil-soluble QDs
(black line), water-soluble AMP-QDs after phase transfer (red
line), and the recovered QDs (blue line).
[0014] FIGS. 9A-B--show luminescence stability of AMP-QDs in PBS
buffers with different pH values. (A) Temporal evolution of
relative PL intensities of AMP-QDs samples under different pH
values. (B) Luminescence images of AMP-QDs samples with different
pH values under UV light irradiation after stored for 30 days.
[0015] FIGS. 10A-B--show colloidal stability of AMP-QDs in NaCl
solutions with different concentrations. (A) UV-vis spectra of
AMP-QDs in NaCl solutions for one day, and (B) temporal evolution
of relative PL intensities of AMP-QDs in NaCl solutions.
[0016] FIG. 11--is a graph showing temporal evolution of
photo-luminscence (PL) intensity of AMP-capped QDs under the
irradiation by a UV lamp at 254 nm excitation together with a
reference sample MPA-QDs.
[0017] FIG. 12 is a graph showing temporal evolution of PL peak
intensities of AMP-QDs and referenced MPA-QDs in the process of
heating at 100.degree. C. The first measurement point corresponds
to samples at room temperature.
[0018] FIG. 13A-B--are representative images of HO-8910 cancer
cells (A) and SMMC-7721 cancer cells (B) incubated with
adenosine-QDs under different, incubation times.
DETAILED DESCRIPTION
[0019] The ability to produce reversibly water-soluble and
biocompatible nanocrystals (NCs) that preserve their detectible
signal (e.g. high luminescent brightness), while maintaining
colloidal and chemical stability, is of great importance for
biological applications. Several strategies have been previously
described for reversibly transferring NCs between hydrophobic and
hydrophilic media. These include using weak surface-binding
molecules, or using stimulus responsive ligands, such as electric
field-, temperature-, or pH-sensitive ligands, and ligands that can
undergo reversible host-guest complexation. Unfortunately, all
reported reversible phase transfer approaches suffer from one or
more of the following problems: i) the phase transfer process will
deteriorate physicochemical properties of NCs, such as colloidal
stability and optical properties; ii) the methods only work for
certain NC systems; iii) NCs may be dispersed in either polar or
nonpolar solvent, but not truly reversible between aqueous and
organic media; iv) the phase transfer reagents require tedious
synthesis and are expensive.
[0020] Presently disclosed is a general ligand-exchange methodology
using nucleotides and/or nucleosides to replace the initial
hydrophobic ligands (with long hydrocarbon tails) on the surface of
NCs, thus rendering the initial oil-soluble NCs reversibly
water-soluble. A reversibly water-soluble nanocrystal comprising a
core nanocrystal capped by a plurality of nucleotides, a plurality
of nucleosides, or a combination thereof is provided. The disclosed
methodology is suitable for a variety of NC systems such as
semiconductor, metal oxide, and to noble metal of different sizes
and shapes. The method may, for example, be employed with
luminescent quantum dots (QDs), since rendering the as-synthesized
oil-soluble QDs water-soluble and biocompatible, while preserving
the high luminescent brightness and maintaining colloidal and
chemical stability, is of great importance for biological
applications. The disclosed nucleotides- and/or nucleosides-capped
QDs, after phase transfer, endow QDs with excellent fluorescent and
colloidal stability independent on the pH and ionic strength,
minimal hydrodynamic size, and are thus suitable potential
fluorophores in biomedical imaging applications. The disclosed
reversibly water-soluble NCs can be readily further functionalized
by hydrophobic ligands via a secondary ligand-exchange reaction,
allowing fully reversible phase transfer and surface
functionalization of NCs.
[0021] The NCs used were synthesized following standard protocols
(as further described in the Examples) with use of capping ligands
(mainly oleic acid and OAm) containing long hydrocarbon tails to
render NCs stable and dispersible in hydrophobic media. For a
typical ligands exchange procedure, a nucleotide or nucleoside
solution in ethanol was added into the purified organics-capped NCs
in a nonpolar solvent (such as hexane, chloroform). The two-phase
mixture containing immiscible layers of ethanol and hexane was
vigorously stirred for about 30 min, after which deionized water
was added into the solution and the NCs was completely transferred
from organic layer to aqueous phase. A series of nucleotide
monophosphates (RNA monomer) including AMP, GMP, CMP and their
corresponding nucleosides have been proven to possess this
capability to render the initial organic ligands capped oil-soluble
NCs water-soluble. Significantly, this phase transfer strategy can
be successfully applied to NCs having a variety of compositions,
sizes, and shapes, including semiconductor NCs such as CdSe/CdS/ZnS
core/shell QDs (6.0 nm), Bi.sub.2S.sub.3 nanorods (8 nm.times.46
nm, FIG. 2B), metal oxides such as cube-shaped MnO nanostructures
(23 nm, FIG. 2C), noble metal Au nanodots (7.5 nm, FIG. 2D), as a
general avenue to obtain water-soluble NCs (see Figures). From the
TEM images, it was found that size, morphology, or size
distribution of the NCs has no observable alteration before and
after phase transfer. In particular, the resulting
nucleotides-capped NCs dispersions in water are stable for several
months without any detectable aggregation or precipitation. Thus,
this method provides a general and facile strategy for decorating
hydrophobic NCs of various materials with hydrophilic ligands
without changing their morphology and composition.
[0022] In one aspect, there is provided a reversibly water-soluble
nanocrystal comprising a core nanocrystal capped by a plurality of
nucleotides, a plurality of nucleosides or a combination thereof.
The core nanocrystal may comprise a quantum dot, a nanorod, a metal
is oxide nanocube, or a noble metal nanodot. The core nanocrystal
may be a quantum dot having a core/shell/shell structure, such as
CdSe/CdS/ZnS. The core nanocrystal may be a nanorod, such as
Bi.sub.2S.sub.3. The core nanocrystal may be a nanocube, such as
MnO, or may be a gold nanodot. Methods of synthesizing nanocrystals
are well known in the art (see, e.g., Qu et al., J. Am, Chem. Soc.
(2002) 124: 2049; Li et. al., J. Am. Chem. Soc. (2003) 125: 12567;
Xie et al., J. Am. Chem. Soc. (2005) 127: 7480).
[0023] The plurality of nucleotides may comprise adenosine
5'-monophosphate, guanosine 5'-monophosphate, cytidine
5'-monophosphate, uridine 5'-monophosphate, thymidine
5'-monophosphate, or combinations thereof. The plurality of
nucleosides may comprises adenosine, guanosine, cytidine, uridine,
thymidine, inosine, or combinations thereof. Alternatively, the
plurality of nucleotides may comprise adenosine 5'-diphosphate,
guanosine 5'-diphosphate, cytidine 5'-diphosphate, uridine
5'-diphosphate, thymidine 5'-diphosphate, adenosine
5'-triphosphate, guanosine 5'-triphosphate, cytidine
5'-triphosphate, uridine 5'-triphosphate, thymidine
5'-triphosphate, or combinations thereof. The plurality of
nucleotides may also alternatively comprise deoxynucelotides and
the plurality of nucleosides may also alternatively comprise
deoxynucleosides.
[0024] Nucleotide and nucleosides (including their mono-, di-, and
tri-phosphate forms as well as deoxy- forms) are extensively well
known, are commercially available from a variety of vendors (e.g.
Sigma Chemical), and are cheap raw material for commercial use.
They alternatively may be synthesized by methods well known in the
art.
[0025] The disclosed reversibly water-soluble nanocrystal may be
further characterized by being soluble and stable in water for at
least about sixty days without substantial aggregation or
precipitation. It may be further characterized by retaining
substantially the same size, morphology, and size distribution when
dispersed in water as compared to the core nanocrystal when capped
by hydrophobic ligands and dispersed in hydrophobic media. It may
be further characterized by retaining substantially the same
luminescence brightness and colloidal stability when dispersed in
water as when dispersed in a hydrophobic media.
[0026] The disclosed reversibly water-soluble nanocrystal may be
further characterized in that the plurality of nucleotides, the
plurality of nucleosides or the combination thereof that have
capped the core nanocrystal form a layer that is equal to or less
than about five nanometers thick. It may be further characterized
in that the nanocrystal does not comprise any hydrophobic ligands.
It may be still further characterized by being soluble in a
water-miscible alcohol. The alcohol may be methanol, ethanol,
ethylene glycol, or combinations thereof.
[0027] The reversibly water-soluble nanocrystal may be further
characterized in that the water-solubility may be reversed by
displacing the plurality of nucleotides and/or nucleotides that
have capped the core nanocrystal with a plurality of hydrophobic
capping ligands. Such reversal of the water-solubility may be
accomplished at room temperature in one hour or less. The
conversion between water-solubility and water-insolubility may be
carried out for at least ten cycles without significantly altering
the performance characteristics of the nanocrystal.
[0028] The reversibly water-soluble nanocrystal may further be
characterized by retaining substantially the same luminescence
brightness and colloidal stability when dispersed in aqueous media
having a pH in the range of three to thirteen as when dispersed in
aqueous media of neutral pH. It may further be characterized by
retaining substantially the same luminescence brightness and
colloidal stability when dispersed in aqueous media having a salt
concentration in the range of one molar to five molar as when
dispersed in aqueous media having a salt concentration of zero
molar.
[0029] The reversibly water-soluble nanocrystal may be further
characterized by having superior resistance to
photo-oxidation-induced precipitation in aqueous media when
compared to a similar nanocrystal capped with mercaptopropionic
acid in the same aqueous media. It may further be characterized by
the core nanocrystal being a quantum dot, and wherein the superior
resistance to photo-oxidation-induced precipitation is measured as
retaining to substantially the same luminescence brightness after
one hour exposure to 6-watt, 254 nm UV irradiation as prior to such
exposure.
[0030] The reversibly water-soluble nanocrystal may further be
characterized by having superior resistance to temperature-induced
precipitation in aqueous media when compared to a similar
nanocrystal capped with mercaptopropionic acid in the same aqueous
media.
[0031] It may further be characterized by the core nanocrystal
being a quantum dot, and wherein the superior resistance to
temperature-induced precipitation is measured as retaining
substantially the same luminescence brightness after one-half hour
at one hundred degrees Celsius as when at room temperature.
[0032] The disclosed reversibly water-soluble nanocrystal may be
further characterized by retaining substantially the same
luminescence brightness and colloidal stability within a biological
cell when compared to outside a cell in aqueous media.
[0033] In another aspect, there is provided a nanocrystal
composition comprising a plurality of reversibly water-soluble,
nucleotide- and/or nucleoside-capped nanocrystals, wherein the
nanocrystals:
[0034] (i) are soluble and stable in water for at least two months
without any substantial aggregation or precipitation;
[0035] (ii) retain substantially the same size, morphology, size
distribution, and performance characteristics when dispersed in
water as compared to a similar nanocrystal capped by hydrophobic
ligands and dispersed in hydrophobic media; and
[0036] (iii) may be rendered reversibly water-insoluble by
displacing the nucleotides and/or nucleosides that cap the
nanocrystals with a plurality of hydrophobic capping ligands.
[0037] In another aspect, there is provided a method for producing
reversibly water-soluble nanocrystals, the method comprising the
steps of:
[0038] (i) providing water insoluble nanocrystals comprising core
nanocrystals capped by a plurality of initial hydrophobic ligands;
and
[0039] (ii) contacting the water insoluble nanocrystals of step (i)
with a plurality of nucleotides, a plurality of nucleosides, or a
combination thereof, thereby replacing the initial hydrophobic
ligands that cap the core nanocrystal with a cap of nucleotides,
nucleosides, or a combination thereof, thus rendering the core
nanocrystal reversibly water-soluble.
[0040] In another aspect, there is provided a method for reversibly
transferring nanocrystals between a hydrophobic solution and a
hydrophilic solution, the method comprising:
[0041] (i) providing a hydrophobic solution comprising core
nanocrystals capped by a plurality of hydrophobic ligands;
[0042] (ii) mixing the hydrophobic solution with a solution
comprising a plurality of nucleotides and/or nucleosides in order
to replace the hydrophobic ligands capping the core nanocrystal
with the nucleotides and/or nucleosides, thereby producing a
ligand-exchanged solution comprising nucleotide- and/or
nucleoside-capped nanocrystals; and
[0043] (iii) mixing the ligand-exchanged solution with a
hydrophilic solution, whereby the nucleotide- and/or
nucleoside-capped nanocrystals transfer from the hydrophobic,
ligand-exchanged solution to the hydrophilic solution.
[0044] In the above-described method, the hydrophobic solution may
be hexane. The hydrophilic solution is water, a water-miscible
alcohol, or a combination thereof. The method may further comprise
the step of:
[0045] (iv) mixing the hydrophilic solution with (a) a solution
comprising a plurality of hydrophobic ligands and (b) a secondary
hydrophobic solution in order to replace the nucleotides and/or
nucleosides capping the core nanocrystal with the hydrophobic
ligands, thereby producing a secondary ligand-exchanged solution
comprising hydrophobic ligand-capped nanocrystals, whereby the
hydrophobic ligand-capped nanocrystals transfer from the
hydrophilic solution to the secondary hydrophobic solution.
[0046] The hydrophobic ligands may comprise organic amine ligands,
for example oleylamine, hexadecylamine, dodecylamine, or
combinations thereof. The secondary hydrophobic solution may be
hexane. Organic amine ligands suitable for capping nanocrystals in
hydrophilic media are well known in the art. Similarly, hydrophobic
solutions suitable for conducting extraction of nanocrystals
between hydrophobic and hydrophilic phases are well known in the
art.
[0047] In another aspect, there is provided a composition
comprising a biological reagent coupled to a reversibly
water-soluble nanocrystal comprising a core nanocrystal capped by a
plurality of nucleotides, a plurality of nucleosides, or a
combination thereof. The biological reagent may comprise an
antibody, a nucleic acid probe, an enzyme substrate, a binding
protein, or combinations thereof. Such reagents are widely
commercially available from a number of vendors, and methods for
generating such reagents are well known in the art. For example,
the antibody may specific for phosphorylated epidermal growth
factor receptor (EGFR), and the disclosed composition thus allows
for the nanocrystal-based detection of the binding of such antibody
to phosphorylated EGFR. By way of a second example, the nucleic
acid probe may be specific for a gene translocation mutation, such
as the BCR-ABL translocation in leukemia, and the disclosed
composition thus allows for the nanocrystal-based detection of the
binding of such probe to BCR-ABL.
[0048] In another aspect, there is provided a method for labeling a
biological cell or molecular component of such cell, the method
comprising the step of:
[0049] contacting the cell or molecular component with a reversibly
water-soluble nanocrystal comprising a core nanocrystal capped by a
plurality of nucleotides, a plurality of nucleosides, or a
combination thereof.
[0050] In another aspect, there is provided a method for labeling a
biological cell or molecular component of such cell, the method
comprising the step:
[0051] contacting the cell or molecular component with a
composition comprising a biological reagent coupled to a reversibly
water-soluble nanocrystal comprising a core nanocrystal capped by a
plurality of nucleotides, a plurality of nucleosides, or a
combination thereof.
[0052] In another aspect, there is provided a biological cell or
molecular component of such cell labeled with a reversibly
water-soluble nanocrystal comprising a core nanocrystal capped by a
plurality of nucleotides, a plurality of nucleosides, or a
combination thereof. The biological cell or molecular component of
such cell may be a cancer cell, or other mammalian cell of
interest, such as a neural cell. The molecular component of such
cell may be a nuclear membrane, or other organelle or cell
structure, such as mitochondria.
[0053] In another aspect, there is provided a biological cell or
molecular component of such cell labeled with a composition
comprising a biological reagent coupled to a reversibly
water-soluble nanocrystal comprising a core nanocrystal capped by a
plurality of nucleotides, a plurality of nucleosides, or a
combination thereof.
[0054] The following Examples are provided only to further
illustrate the disclosure, and are not intended to limit its scope,
except as provided in the claims appended hereto. The present
disclosure encompasses modifications and variations of the methods
taught herein which would be obvious to one of ordinary skill in
the art.
EXAMPLE
Synthesis of Oil-Soluble Nanocrystals
[0055] In these Examples, adenosine 5'-monophosphate (AMP),
adenosine, adenine, guanosine 5'-monophosphate (GNP), guanosine,
cytidine 5'-monophosphate (CMP), and cytidine were purchased from
Sigma. (MPA, >99%), tetramethylammonium hydroxide pentahydrate
(TMAH, >95%), oleylamine (OAm, 70%), 1-hexadecylamine (98%),
1-dodecylamine (>99%), oleic acid (99%), 1-octadecene (ODE,
>95%) were obtained from Aldrich and used without further
purification. All organic solvents such as hexane, dichloromethane,
ethanol, methanol were of analytical grade and obtained from
commercial sources and used as received. Deionized water was used
throughout.
[0056] A. CdSc/CdS/ZnS Core/Shell/Shell Quantum Dots (QDs).
[0057] CdSe core nanocrystals were prepared via a modified
literature method (see, e.g., Qu et al., J. Am. Chem. Soc. (2002),
124: 2049. Typically, 25.6 mg (0.2 mmol) of CdO, 1.2 g of TOPO, 1.0
ml of oleic acid and 4.0 ml of ODE were loaded in a 50 mL
three-neck flask clamped in a heating mantle. The mixture was
heated to 320-330.degree. C. under argon flow and resulted in a
colorless clear solution, which was then cooled to 315.degree. C.
At this temperature, 2.4 mL of the Sc precursor solution, which was
made by dissolving selenium (79.0 mg) in TOP (4.0 ml) and ODE (6.0
ml) by sonication, was quickly injected into the reaction flask.
After the injection, the reaction temperature was set at .about.270
.degree. C. for the growth of the nanocrystals with different
periods of time (10 s-3 min) to get nanocrystals with desired size.
The reaction mixture was then allowed to cooled to
.about.60.degree. C. and 10.0 ml of hexane/CH.sub.3OH (v/v, 1:1)
was used as the extraction solvent to separate the nanocrystals
from byproducts and unreacted precursors. The as-prepared CdSe
solution was further purification by centrifugation and decantation
with the addition of acetone and then the CdSe NPs were redispersed
in hexane.
[0058] Stock solutions preparation. The Zn precursor solution (0.1
M) was prepared by dissolving 219.5 mg (1 mmol)
Zn(OAc).sub.2.2H.sub.2O in 10.0 mL ODE at 160.degree. C. The sulfur
precursor solution (0.1 M) was obtained by dissolving sulfur in ODE
at 120.degree. C. The Cd precursor solution (0.1 M) was prepared by
dissolving 128.4 mg (1 mmol) CdO in 2.0 ml oleic acid and 8.0 mL
ODE at 160.degree. C. Each stock solution was stored at room
temperature. Synthesis of CdSe/CdS/ZnS core/Shell/Shell QDs. The
successive ion layer adsorption and reaction (SILAR) technique was
adopted for the growth of CdSe/CdS/ZnS core/shell/shell
nanocrystals (see, e.g. Li et al., J. Am. Chem. Soc. (2003),
125:12567; Xie et al., J. Am. Chem. Soc. (2005), 127: 7480. In a
typical procedure, a chloroform solution of purified 3.5 nm CdSe
QDs containing 0.1 mmol of CdSe, 1.0 mL of oleylamine and 4.0 mL of
ODE were loaded in a 50 mL flask. The flask was then pumped down at
room temperature for 20 min to remove the chloroform and at
100.degree. C. for another 20 min while flushing the reaction
system twice with a flow of argon. Subsequently, the reaction
mixture was further heated to 230.degree. C. for the overgrowth of
the CdS shell. The Cd precursor stock solution was added into the
reaction flask, after 10 min when the Cd precursor was fully
deposited around the CdSe surface, an equimolar amount of S
precursor stock solution was added into the reaction system. When
the first monolayer of CdS was deposited around the CdSe cores,
another Cd/S precursor solution was added alternately at
approximately 10 min intervals. The volume of the precursor stock
solution added in each cycle was the amount needed for a whole
monolayer of CdS shell. The amount was calculated from the
respective volumes of concentric spherical shells with 0.35 nm
thickness for one monolayer (ML) of CdS (e.g. 0.7, 1.0, 1.3 mL for
the 1.sup.st , 2.sup.nd, and 3.sup.rd ML, respectively). Then the
reaction temperature was set at 200.degree. C. for the overgrowth
of ZnS shell. The Zn/S precursor stock solution was added into the
reaction flask at intervals of 20 min. To monitor the reaction,
aliquots were taken before a new cycle of injection and their
corresponding UV-vis and PL spectra were recorded. The reaction was
terminated by allowing the reaction mixture to cool down to room
temperature. The purification procedure was similar to that for
CdSe core nanocrystals.
[0059] B. Synthesis of Bi.sub.2S.sub.3 Nanorods.
[0060] Nanorods were produced essentially according to standard
methods (see, e.g. Wu, et al., Nano Res. (2010), 3: 379-386). 31.5
mg (0.1 mmol) of BiCl3 powder was added to a flask containing 2.0
mL of OAm followed by degassing at 70.degree. C. for 5 min under
vacuum to remove the moisture and oxygen. The reaction vessel was
then filled with nitrogen and the temperature was increased to
150.degree. C. with a heating rate of 10.degree. C./min and the
vessel maintained at this temperature until the complete
dissolution of BiCl3 powder to give a white milky solution. Then
2.0 mL of OAm containing 11.3 mg (0.15 mmol) of thioacetamide was
injected into the reaction system. After the addition of
thioacetamide, the temperature of the reaction system was further
increased to 180.degree. C. and the vessel maintained at this
temperature for 5-10 min. The reaction mixture was cooled to room
temperature and the resulting nanostructures were precipitated by
anhydrous ethanol, The Bi2S3 NPs were redispersed in hexane to give
a brown dispersion.
[0061] C. Synthesis of MnO Nanocubes
[0062] Nanocubes were produced essentially according to standard
methods (see, e.g., Zhong et al., J. Phys. Chem. B (2006), 110:
2-4; Yin et al., J. Am. Chem. Soc. (2003), 125: 10180-10181). 24.5
mg (0.1 mmol) of Mn(OAc)2 and 2 mL of oleylamine were mixed with 5
mL ODE in a 50 mL flask. Under nitrogen flow, the mixture was
quickly heated to 130.degree. C. under magnetic stirring. The
formed solution was kept at this temperature for 10 min and cooled
down to room temperature. Then 30 mL of anhydrous ethanol was added
into the solution, and the suspension was centrifuged at 4000 rpm
for 5 min. The supernatant was discarded and the MnO NPs were
redispersed in hexane to give a brown dispersion.
[0063] D. Synthesis of Au Nanodots.
[0064] Gold nanodots were produced essentially accordingly to
standard methods (see, e.g., Shen et al., Chem. Mater, (2008), 20:
6939-6944). 34.0 mg (0.1 mmol) of HAuCl.sub.43H.sub.2O and 1.0 mL
of oleylamine were mixed with 15 mL toluene in a 50 mL flask. Under
nitrogen flow, the mixture was slowly heated to 65.degree. C. under
magnetic stirring. The formed solution was kept at this temperature
for 6 h and cooled down to room temperature. Then 30 mL of
anhydrous ethanol was added into the solution, and the suspension
was centrifuged at 4000 rpm for 5 min. The supernatant was
discarded and the Au NPs were redispersed in hexane to give a red
dispersion.
[0065] FIG. 1 shows TEM images of initial oil-soluble NCs before
phase transfer: (A) CdSe/CdS/ZnS QDs, (B) Bi.sub.2S.sub.3 nanorods,
(C) MnO nano-cubes, and (D) Au nanodots. Typical morphology, size,
and dispersion of each type of exemplary water-insoluble
nanocrystal is shown.
EXAMPLE 2
[0066] Production of Water-Soluble Nanocrystals
[0067] Exchange of the native hydrophobic ligands on QDs surface
(as produced in Example 1A) by AMP (or other nucleotide or
nucleoside ligands including adenosine, adenine, GMP, guanosine,
CMP, and cytidine) was performed as follows. Typically, 1.0 g of
AMP was dissolved in 3.0 mL of ethanol and the pH of the resulting
solution was adjusted to 10 with the use of concentrated NaOH or
TMAH solution. Then 0.3 mL of the obtained AMP solution in ethanol
was added dropwise into a purified QDs solution in hexane (or
CHCl.sub.3) (10.sup.-6 M, 20.0 mL), and vigorously stirred for 30
min. Subsequently, deionized water was added into the solution. The
QDs were found to be successfully transferred from the hexane phase
on the top to the water phase in the bottom. The colorless organic
phase was discarded and the aqueous phase containing the QDs was
collected. The excess amount of free ligand was removed by
centrifugation purification with use of acetone. The supernatant
was discarded and the pellet was then re-dissolved in water and
repeated this centrifugation-decantation process three times to get
the purified QDs aqueous solutions.
[0068] FIG. 2 shows luminescence images of initial oil-soluble QDs
in hexane, and alcohol-soluble QDs dispersed in methanol and
ethylene glycol (EG) after phase transfer with use of AMP
lamination by room light (A) and UV light (B). The images show that
phase-transfer of nucleotide/nucleoside-capped core nanocrystals
has occurred, and these capped nanocrystals are dispersed in the
aqueous (water-miscible MeOH) phase.
[0069] FIG. 3 shows TEM images AMP-modified NCs dispersed in water
derived from the initial oil-soluble ones via phase transfer; (A)
CdSe/CdS/ZnS QDs (6.0 nm), (B) Bi.sub.2S.sub.3 nanorods (8
nm.times.46 nm), (C) MnO nano-cubes (23 nm), and (D) Au nanodots
(7.5 nm). The insets show photographs of the initial OAm-capped NCs
dispersed in hexane (left) and the resulting AMP-capped NCs
dispersed in water (right) after phase transfer. The images
indicate that nucleotide/nucleoside-capped, water-soluble
nanocrystals maintain essentially the same size, morphology, and
distribution in aqueous phase as the initial, equivalent
hydrophobic ligand-capped core nanocrystals in oil phase.
[0070] In these Examples, UV-vis and PL spectra were obtained on a
Shimadzu UV-2450 spectrophotometer and a Cary Eclipse (Varian)
fluorescence spectrophotometer, respectively. FT-IR spectra were
measured with samples pressed into a KBr plate and recorded from
4000 to 400 cm.sup.-1 using a Nicolet Magna-IR 550 FT-IR
spectrometer. Transmission electron microscopy (TEM) images were
taken on a JEOL JEM-1400 at an acceleration voltage of 100 kV.
Dynamic light scattering (DLS) was conducted with a Zeta Sizer nano
series laser light scattering system (Malvern Instrument
Corporation). Confocal fluorescence imaging was performed with an
OLYMPUS ZX81 laser scanning microscopy and a 60.times.
oil-immersion objective lens.
EXAMPLE 3
Characteristics of Nucleotide/Nucleoside-Capped Nanocrystals
[0071] The phase transfer experiments of CdSe/CdS/ZnS core/shell
QDs were performed on three typical emission wavelengths of 552,
594, and 626 nm. The selected nucleotide monophosphates and
nucleosides all showed superior performance for the phase transfer
of the organic ligands capped hydrophobic QDs into water-soluble
ones. As a demonstration, FIG. 4 shows the luminescence images of
three QDs samples before and after phase transfer with use of AMP
and adenosine, respectively. No distinguishable luminescence
brightness of the QDs before and after phase transfer with use of
either AMP or adenosine, demonstrating the superior performance of
both AMP and adenosine in phase transfer of oil-soluble QDs. The
phase transfer process was facile and occurred with nearly 100%
efficiency, which was determined by observing almost no
luminescence emission and no absorption corresponding to QDs in the
optical spectra of the organic phase after the phase transfer.
[0072] After phase transfer, the AMP-capped QDs aqueous solutions
exhibited identical absorption and PL emission spectral profiles to
those of original hydrophobic QDs in hexane (see FIG. 3). This
indicates that the surface ligands have no effects on the
electronic properties of the inorganic QDs cores and no aggregation
or surface degradation of the QDs occurred upon the process of
phase transfer. The original high florescent brightness was
preserved for all selected QDs samples with different emission
colors after the phase transfer and the water-soluble AMP-capped
QDs show almost identical PL QYs in the range of 50-65% as those of
the original oil-soluble ones. It should be noted that heavy loss
of luminescence brightness of QDs after phase transfer into aqueous
solutions was commonly observed in previously reported attempts to
generate water-soluble nanocrystals.
[0073] FIG. 4 shows TEM images of 6.0 nm QDs with emission
wavelength of 594 nm before and after phase transfer with use of
AMP as phase transfer reagent, respectively. TEM measurements
showed that the AMP-capped QDs provided well-dispersed
distributions of nanoparticles. They were well isolated from each
other and no aggregation or clumping occurred, revealing that the
NCs size and shape are preserved. The aggregate-free nature of the
QDs dispersions in water is further verified by the monomodal size
distributions measured by DLS as shown in the inset of FIG. 4B. The
average hydrodynamic diameter (HD) of AMP-QDs extracted from the
DLS measurement results was about 7.1 nm, which is only a little
larger than the size observed by TEM (6.0 nm). The thickness of the
capping layer AMP is deduced as only about 1.1 nm. This compact
shell matches the multi-dentate coordination mode of AMP ligand on
the QDs surface as described below. The observed HD of these
AMP-capped QDs is remarkably smaller than those of amphiphilic
polymer coated QDs, which have HDs on the level of 20-50 nm. The
reduced HD of water-soluble luminescent QDs is important for cell
labeling and single-particle tracking applications. These above
features suggest that nucleotides ligands benefit the surface
stabilization of QDs for their high fluorescence QYs, well
monodispersibility, and remarkably small HD.
[0074] The fact that phase transfer was achieved via the
ligand-exchange was confirmed by FTIR spectroscopy. The FTIR
spectra of the initial OAm-capped QDs (FIG. 6) show the
characteristic C--H stretching vibrations, derived from long
hydrocarbon chain portion in OAm molecules, at 2800-3000 cm.sup.-1
with strong intensity; while those of water-soluble QDs show almost
no signals in the C--H stretching vibration region. This indicates
that the original OAm molecules attached to the QDs surface were
near completely removed after phase transfer. It was found that the
spectra of water-soluble QDs after phase transfer (FIG. 6B) show a
similar spectral profile to those of free Na.sub.2-AMP (FIG. 6C).
Thus it can be concluded that the capping ligand on QDs surface has
been changed to AMP after this phase transfer. In the spectrum for
AMP-QDs, the broad band around 3500 cm.sup.-1 is assigned to the
O--H vibration of solvated water molecules and the OH group from
the sugar portion of AMP. The presence of solvated water molecules
is consistent with the hydrophilic nature of the AMP-QDs. Due to
the coordination of AMP molecules on the surface of QDs, most of
the sharp vibration in the spectra of free AMP molecules are
broadened in those of AMP-QDs and the broadening phenomenon for
capped ligands on surface of nanoparticles has been commonly
observed in previous literature reports. The features associated
with the vibration of phosphate group (cm 950-1100 cm.sup.-1) and
the purine ring (ca. 1450-1650 cm.sup.-1) of the nucleotides were
all broadened in the AMP-QDs. The FTIR spectroscopy analysis
confirms that the initial OAm capping molecules are indeed replaced
by AMP from the surface of QDs in the process of phase
transfer.
[0075] The facts that adenosine molecule without the existence of
phosphate group can also render the initial oil-soluble QDs
water-soluble via ligand exchange can deduce that the purine group
of AMP or adenosine is evolved into the coordination with the
surface metal atoms of QDs. Furthermore, the small molecule adenine
is a model compound to perform the phase transfer. Similarly,
adenine can also render the initial oil-soluble OAm-QDs
water-soluble as done by AMP or adenosine. This further confirms
that purine ring indeed coordinate to the nanoparticles.
[0076] The disclosed nucleotides- and/or nucleosides-capped QDs
obtained through phase transfer can disperse in water-soluble
alcohols such as methanol, ethanol, and ethylene glycol (EG).
Images of emissions color of initial oil-soluble QDs dispersed
hexanes and AMP-capped QDs dispersed in MeOH and EC after ligand
exchange are shown in FIG. 2. Since ethanol and hexane are partly
miscible and no clear boundary can be formed, the photograph of QDs
dispersed in ethanol was not given. Similar to the case of QDs
dispersed in aqueous media, the MPA-QDs dispersed in alcohol media
also preserve the high fluorescent brightness and spectral profile.
It should be noted that the access of high quality (high PL QY and
high colloidal stability) alcohol-dispersible QDs is even more
difficult than preparation of water-soluble luminescent QDs and
thus few tenable approaches have previously been reported. In
addition, the access of alcohol-soluble NCs plays the crucial role
for the silica coating process via stober route. It is not
unexpected for the alcohol-soluble of the nucleotides capped QDs
since multiple hydroxyl groups existing in the nucleotide
molecules.
EXAMPLE 4
Reversible Phase Transfer of Water-Soluble Nanocrystals
[0077] A significant benefit of the phase transfer method disclosed
herein is that the resulting water- and alcohol-soluble
nucleotides- and/or nucleotides-modified nanocrystals can be
further transferred back to oil-soluble with use of hydrophobic
ligands. This allows for reversible phase transfer and further
surface functionalization of NCs. Upon the addition of organic
amine ligands such as OAm, hexadecylamie, or dodecylamine to the
nucleotides-capped QDs dispersion in water or alcohol combined with
hexane, QDs were found to transfer completely from the water or
alcohol layer to the hexane layer after stirring for about 10
minutes, suggesting that QDs are successfully capped by hydrophobic
amine ligands. The FTIR spectrum of the OAm-capped QDs recovered
from the water-soluble AMP-Ws are shown in FIG. 7, which displays
the characteristic C--H stretching vibration signals in the range
of 2800-3000 cm.sup.-1.
[0078] After transfer back to oil-phase, the hydrophobic QDs where
purified by centrifugation to remove excess amount of free amine
ligands, and again, adding nucleotide ligands to the recovered QDs
solution in hexane following the previous phase transfer step, QDs
will be rendered to water-soluble again. This completely reversible
process can be performed at room temperature, providing a feasible
method to obtain hydrophobic nanoparticles from aqueous medium, or
vice versa. FIG. 8A illustrates the reversible phase transfer
results performed on CdSe/CdSe/ZnS QDs. When oil-soluble QDs were
transferred into water-soluble ones, the decrease of PL brightness
is less than 5%; while the slightly decreased PL brightness can be
recovered almost completely after transferred back into
oil-soluble. It should be highlighted that the transfer between
aqueous and oil phases can be repeated for more than 10 cycles
without significantly altering the fluorescent brightness. FIG. 8B
shows the variation of relative PL intensities of oil- and
water-soluble QDs in each phase transfer cycle and the
representative PL emission spectra are shown in FIG. 8C. This
facilely reversible surface modification is attributed to the weak
binding affinity of hydroxyl portion in nucleotides/nucleosides to
the NC surface.
EXAMPLE 5
Stability Water-Soluble Nanocrystals
[0079] A. pH Stability,
[0080] To test pH stability of the water-soluble nanocrystals, 100
.mu.L of purified concentrated AMP-QDs aqueous solution was added
and mixed well into a 4.9 mL of PBS buffer solution with different
pH values (different pH values were obtained with the addition of
HCl or NaOH solution to the 50 mM phosphate buffer with initial pH
of 7.0). The obtained AMP-QD solutions with various pH values were
sealed and stored in the stark and monitored their PL spectra over
time. By this procedure, the QDs solutions with different pH values
were of identical concentration in the testing period. Since the PL
spectra of all samples were measured under identical instrumental
conditions, the PL intensities of the corresponding QDs can
represent their corresponding QYs and can be used to compare their
pH sensitivity.
[0081] In FIG. 9A, the temporal evolution of PL intensities of
AMP-capped QDs in representative pH buffer solutions were
presented. As expected, the obtained AMP-coated QDs can remain high
PL QYs and colloidal stability, aggregation-free over extended
periods of time (months) in a broad pH range (3-13). It was
observed that in the pH range of 3-13, highly fluorescent
brightness can be preserved without significant luminescence loss
(<10%) in a time period of 30 days. In both the extremely low pH
of 2 and extremely high pH of 14, the PL intensity decreased in a
relative fast fashion. After 15 days, the corresponding PL
intensity can only retain 60% and 19%, respectively. The emission
colors of AMP-capped QDs after staying for 30 days were shown in
FIG. 9B. Except the samples at pH 1 or 14, all the samples showed
bright luminescence without observed precipitation. This reflects
highly stable property of their luminescence and superior
dispersibility in a wide pH range. In comparison,
thiol-functionalized carboxylic acid (such as MPA, DHLA) capped QDs
can only keep high PL QY and colloidal stability at neutral to weak
basic solutions as reported in previous literature, since a high pH
value is favor for deprotonation of carboxylic acids and
deprotonation of carboxyl groups is known to be the crucial factor
to solubilize these carboxylated ligand coated QDs. This narrow pH
range limits their potential biomedical application.
[0082] The superior colloidal and luminescent stability of the
disclosed nucleotides/nucleosides-capped QDs may stem from the
intrinsic molecular structure of nucleotides as proven in the
nucleotides capped CdS QDs synthesized directly in aqueous media.
As an instance, AMP is a compound with three hydrophilic groups, a
phosphate group, two hydroxyl groups, and endocyclic and exocylic
amine groups. Phosphoric acid is a strong acid with high
pk.sub..alpha. value which is beneficial for the solubility in
strong acidic environment and hydroxyl groups are non-ionic which
is not sensitive to pH values and amine group is prone to
protonation at acidic condition. For the same reason, our
experimental results indicated that adenosine-capped QDs is also
stable a pH 3-13. The colloidal and luminescent stability of
nucleotides-QDs in acidic media suggests that they may be good
candidates as intracellular imaging probes for QD applications
because most intracellular organelles such as endosomes and
lysosomes are acidic (pH 4-6).
[0083] B. Salt Stability.
[0084] The use of QDs in any sensing scheme requires that they
exhibit long-term stability in solutions that span a wide range of
electrolyte concentrations. The aggregation of QDs at elevated salt
concentration may define the limitations of some biological
applications in high ionic strength media, such as intracellular
and in vivo studies, where the ionic concentration is known to be
high. To test the colloidal stability of the nucleotides-QDs in
electrolyte solutions, purified concentrated AMP-QDs solution was
diluted into NaCl solutions with a series of different
concentrations.
[0085] FIG. 10A shows the UV-vis absorption spectra of AMP-QDs
incubated in NaCl solution with different concentrations for a
whole day. For all the absorption spectra, up to the nearly
saturated NaCl solution media (5.0 M), the baselines were all
horizontal and no absorption tail at the long wavelength side was
observed. This indicates that scatter light forum the colloidal
dispersions did not exist and thus no aggregation occurred for the
QDs dispersions throughout the whole NaCl concentration range. It
is noted that the AMP-coated QDs solutions with different NaCl
concentrations could still keep homogeneously dispersible after
stored for more than 30 days. The instant PL spectra of AMP-coated
QDs dispersed in different concentrations NaCl solutions kept a
similar profile but with slightly variation of PL intensities (less
than 3%) as shown in the first measurement points in each curves of
FIG. 10B. During a period of 30 days, the PL intensity of all
samples did not show significant quenching although some
fluctuation was observed within 10% relative intensity variation.
It is highlighted that the PL QYs have a 10-15% increase in the
first 5-10 days when the QDs dispersed in solutions containing
0-2.0 M NaCl. The increase of PL intensity may due to the light
irradiation effect during the storage stage. Multiple hydroxyl
groups provide AMP-QDs a stable hydration layer, which is little
affected by charged ions. This may give the reason for the
superficial colloidal stability of AMP-QDs under high salinity
condition. It is noteworthy that the extraordinary stability of
disclosed AMP-stabilized QDs in high concentration salt solutions
can be comparable to that of reported QDs functionalized by Tween
derivatives, or zwitterionic ligands. This feature of high salinity
tolerance is of special interest to expand their applications to
biology and biomedicine.
[0086] C. Photo-Oxidation Stability.
[0087] To investigate the photostability of the AMP-QDs in PBS
buffer solution (pH=7), the MPA-capped QDs derived from the same
batch of oil-soluble QDs were used as a reference and irradiated
together with the AMP-QDs by a 6-watt UV light at 254 nm in the
presence of oxygen under identical conditions. The selection of
MPA-capped QDs as a reference is because MPA-QDs are the most
common water-soluble ones derived from phase transfer via ligand
exchange procedure and the obtained MPA-QDs exhibit one of the best
results for water-soluble QDs. The used MPA-QDs were prepared
strictly according to the modified procedure developed by Lee (see
Pong et al., Langmuir (2008), 24: 5270-5276). The temporal
evolution of relative PL intensity of both samples is summarized in
FIG. 11, with initial PL intensity for each sample normalized to
100. It was found that shortly after the onset of irradiation, both
QD samples increased in PL intensity (by a factor of 17-21%). This
photo-enhancement effect was commonly observed in previous reports
and the reason may be due to a surface rearrangement of ligands
and/or photocatalytic annealing of surface atoms to repair defects
and recombination centers that formed during a disordered ligand
exchange. The PL enhancement effect by UV irradiation is much more
instant for MPA-QDs than that for AMP-coated QDs. After approaching
maximum value (4 h for MPA-QDs, and 20 h for AMP-QDs), the PL
intensities in both samples decreased gradually. Within 15 h of UV
light exposure, MPA-QDs precipitated gradually, which was in
accordance with previous reports of photooxidation of the
thiol-containing MPA ligand, resulting, in colloidal instability
and aggregation. The facile oxidization feature of thiol group in
MPA ligands deteriorate the photo-stability of the resulting QDs.
In comparison, AMP-capped QDs also endured photo-bleaching but at a
much slower rate than MPA-coated QDs. The bright luminescence of
the AMP-QDs can be retained up to 60 h, after which the PL
intensity decreased gradually and the QDs precipitated after
irradiation of 75 h. This superior photo-stability of AMP-QDs may
be attributed to the anti-oxidation capability of nucleotide
ligands
[0088] D. Thermal Stability.
[0089] Thermal stability of the disclosed water-soluble,
nucleotide/nucleotide-capped nanocrystals was evaluated by
monitoring the evolution of relative luminescence intensity of both
AMP-QDs and reference sample MPA-QDs in PBS buffer (pH=7) at
100.degree. C. in nitrogen atmosphere. The purified AMP-QDs and
MPA-QDs were loaded in a closed container and heated from room
temperature to 100.degree. C. in a period of 10 min and kept at
this temperature for a certain period to monitor the variation of
luminescence intensity. Timing started when the temperature reached
100.degree. C. Experimental results showed that our AMP-QDs
exhibited excellent thermal stability in boiling water. As shown in
FIG. 12 when the samples were heated to 100.degree. C. from room
temperature, the corresponding PL intensities of AMP-QDs could
preserve their high PL QY at room temperature and maintain this
high brightness for 0.5 h, after then the PL intensity decrease
gently and the PL intensity can retain ca. 70, 43 and 36% of the
initial value at room temperature with heating time of 1, 2, and 3
h, respectively. Furthermore, the AMP-QDs could still keep
homogenously dispersible at solution with the heating time up to 3
hours. While in the reference sample MPA-capped QDs, the PL
intensity decreased much more sharply. When the temperature was
raised from mom temperature to 100.degree. C. (corresponding to
time of 0 min), the PL intensity dropped about 40%. With extending
heating, the MPA-QDs aggregated and precipitated gradually in a
period of 1 h. These findings suggest that the exemplary, disclosed
water-soluble nanocrystals (AMP-QDs) are much superior to MPA-QDs
in the aspect of thermal stability.
EXAMPLE 6
Cell Labeling with Water-Soluble Nanocrystals
[0090] The nucleotides- and nucleosides-capped QDs are all very
stable in cell culture medium without luminescence change or
particle aggregation. To establish the suitability of the disclosed
nanocrystals for biological applications, we examined the cellular
uptake of 604 nm emitting adenosine-capped QDs (at .about.100 nM)
in DMEM cell culture medium to explore their suitability in
intracellular imaging. To keep the reliability, we chose two cell
lines, human ovarian cancer cell (HO-8910) and human liver cancer
cell (SMMC-7721).
[0091] HO-8910 and SMMC-7721 cell lines were cultured overnight
(37.degree. C., 5% CO2) on glass chamber slides in Dulbecco's
Modified Eaglets medium (DMEM) supplemented with 4 mM L-glutamine,
1 mM sodium pyruvate, 1% (v/v) penicillin/streptomycin/actinomycin
D-antibiotic/antimyeotic, and 10% (v/v) heat- inactivated fetal
bovine serum (FBS). QDs were adding into the culture medium (final
concentration was 100 nM) and incubated for a certain time. Then
removing from culture medium, cells were fixed with 2%
paraformaldehyde, washed with PBS, followed by
4',6-diamidino-2-phenylindole dihydrochloride (DAN) staining (18.7
uM) of the nuclear for 5 min, washed with PBS for three times, and
finally immersed in 30% (v/v) glycerol/PBS. Fluorescence images
were recorded with 450-550/405 nm and 550-650/488 nm
emission/excitation for the visualization of DAPI and QDs
respectively.
[0092] FIG. 13 shows the confocal microscopy of QDs endocytosed by
HO-8910 (panel A) at different incubation times and the
corresponding results for SNOW-7721 cells (panel B). Each row of
image panels in FIG. 13 shows representative DIC, 604-nm emitting
QDs (red), DAPI (blue), and the merged fluorescent composite images
(right). For incubation of 12 hours, a substantial intracellular
uptake of QDs took place as indicated by the pronounced
fluorescence intensity measured for the cells. With prolonging
incubation time, QDs maintained inner cells and stayed adjacent to
the cell nucleoli.
[0093] It is notable that the adenosine-capped QDs were highly
concentrated in one pole outside the nucleoli of the two kinds of
cancer cells. This demonstrates the specific binding of QDs to the
nuclear membrane, which is different from the commonly observed
non-specific absorption feature of QDs in previous literature
reports. No morphological and QDs PL brightness changes were
observed even under incubation time up to 72 hours. This
demonstrates that the adenosine-capped QDs were stable in cells and
suitable for in vitro cell labeling, cell tracking, and other
bioimaging applications. These promising features will enhance the
performance of QDs as probes in many biological imaging
applications, such as long-term single-molecule tracking or
simultaneous multicolor imaging. It may also be used in other types
of semiconducting or metal nanoparticles and may benefit in vivo
applications, where small size and stability are crucial parameters
for targeting and renal elimination.
* * * * *